Protein engineering to enable site-specific bioconjugation through catechol-boronate coupling chemistry
Date
2021
Authors
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Publisher
University of Delaware
Abstract
Over the past few decades, the customizability provided to biomolecules through
protein conjugation has enabled a diverse set of applications, which include protein-based therapeutics, biomaterials, and biosensors. However, current chemical
modification techniques suffer from lack of specificity that results in heterogeneous
mixtures, limited time stability in vivo, and perturbations to the protein function.
Synthetic biology researchers have overcome some of the challenges by utilizing
genetic code expansion technology to enable precise formation of protein
conjugates. ☐ In this project, we seek to develop a bio-orthogonal protein conjugation
platform in Escherichia coli (E. coli) based on catechol-boronate coupling
chemistry. This chemistry proceeds in aqueous and mild conditions and can be tuned
to be reversible or irreversible according to the end application. In order to perform
catechol-boronate conjugation in proteins, we have site-specifically inserted
catechol amino acids into proteins using non-standard amino acid (nsAA)
incorporation technique. Although a catechol amino acid: L-3,4-
dihydroxyphenylalanine (DOPA) is a proven candidate for ribosomal incorporation,
an unaddressed challenge is the replacement of tyrosine with DOPA due to the
structural similarity. We have investigated in this thesis whether a DOPA analogue
having extended side chain (C5-DOPA) with lower resemblance to tyrosine can be
site-specifically incorporated into proteins. We have screened a library of published xii
OTSs from different families for specificity and activity on C5-DOPA. Based on
our results top performing candidates will be engineered as next step for improved
specificity to incorporate C5-DOPA at multiple instances in a reporter protein.
Description
Keywords
Protein engineering, Bioconjugation, Catechol-boronate coupling